In cardiac muscle, intracellular Ca²⁺ and Mg²⁺ are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca²⁺] in the SR ([Ca²⁺]L) stimulates the Ca²⁺ release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg²⁺, which has not been regarded as an important regulator of Ca²⁺ release. The effects of luminal Ca²⁺ and Mg²⁺ on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal Ca²⁺ produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal Mg²⁺ was observed, pointing to an important physiological role for luminal Mg²⁺ in cardiac muscle. At diastolic [Ca²⁺]C, luminal Mg²⁺ inhibition was voltage independent, with Ki=45 μM at luminal [Ca²⁺] ([Ca²⁺]L) = 100 μM. Luminal and cytoplasmic Mg²⁺ inhibition was alleviated by increasing [Ca²⁺]L or [Ca²⁺]C. Ca²⁺ and Mg²⁺ on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites. The data were accurately fitted by a model based on a tetrameric RYR structure with four Ca²⁺-sensing mechanisms on each subunit: activating luminal L-site (40-μM affi nity for Mg²⁺ and Ca²⁺), cytoplasmic A-site (1.2 μM for Ca²⁺ and 60 μM for Mg²⁺), inactivating cytoplasmic I₁-site (~10 mM for Ca²⁺ and Mg²⁺), and I₂-site (1.2 μM for Ca²⁺). Activation of three or more subunits will cause channel opening. Mg²⁺ inhibition occurs primarily by Mg²⁺ displacing Ca²⁺ from the L- and A-sites, and Mg²⁺ fails to open the channel. The model predicts that under physiological conditions, SR load–dependent Ca²⁺ release (1) is mainly determined by Ca²⁺ displacement of Mg²⁺ from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.